This invention covers compounds which preferentially inhibit, or bind, one form of a phosphodiesterase isozyme denominated 4 (PDE 4 hereafter) while exhibiting equal or, preferably less binding or inhibition for a second form of the enzyme and are thus useful for treating chronic obstructive pulmonary disease (COPD). These isoenzyme forms, and it is believed they are different forms of non-interconvertible conformations of the same enzyme though this has not been proven, are distinguished by their binding affinity for rolipram, an architypical PDE 4 inhibitor. Rolipram binds with high affinity to a site of one form but with low affinity to the catalytic site of the other. Herein one form is denominated the high affinity rolipram binding site and the other form is identified as the low affinity rolipram binding site. A method for selectively treating chronic obstructive pulmonary disease by inhibiting preferentially the low affinity form of the catalytic site in the PDE 4 isozyme is also disclosed. A method for treating COPD comprising administering a compound preferentially binding to the low affinity binding site is also disclosed. A method for selectively treating exercise induced asthma (EIA) by inhibiting preferentially the low affinity form of the catalytic site in the PDE 4 isozyine is also disclosed.
Cyclic nucleotide phosphodiesterases (PDEs) represent a family of enzymes that hydrolyze the ubiquitous intracellular second messengers, adenosine 3xe2x80x2,5xe2x80x2-monophosphate (cAMP) and guanosine 3xe2x80x2,5xe2x80x2-monophosphate (cGMP) to their corresponding inactive 5xe2x80x2-monophosphate metabolites. At least nine distinct classes of PDE isozymes are believed to exist, each possessing unique physical and kinetic characteristics and each representing a product of a different gene family. These have been distinguished using the numerals 1 through 9.
The target enzyme in this invention is the PDE 4 isozyme in all its various forms and in the full domain of its distributions in all cells. It is a low Km (cAMP Km=1-5 xcexcM) cAMP-selective enzyme that has little activity against cGMP (Km greater than 100 xcexcM). Members of this isozyme class have the interesting characteristics of existing in two or more non-interconvertible or slowly interconvertible forms that bind rolipram and other PDE 4 inhibitors with distinct rank order potencies. Thus the same gene product can exist in more than one catalytically active conformational state. Importantly, the relative proportions of the different binding forms may vary depending on the tissue cell type. For example, inflammatory cells may contain a relatively high proportion of the form that binds rolipram with a low affinity while brain and parietal cells may contain a relatively high proportion of the form that binds rolipram with a high affinity.
Of particular interest in this invention is the role this class of isozymes play in inflammation and airway smooth muscle. Studies indicate that it plays a prominent role in regulating cAMP in a wide variety of inflammatory cells (i.e., mast cells. basophils, eosinophils, neutrophils, and monocytes) and airway smooth muscle. The work of this invention is particularly applicable to inflammatory cells and airway smooth muscle; the isozyme type expressed in human monocytes is of particular interest. This is because cyclic AMP serves as a second messenger to inhibit chemotaxis and activation of inflammatory cells. In addition, cAMP mediates smooth airway muscle relaxation. This coupled with the major role of PDE 4 in metabolizing cAMP has provided the underpinnings for investigating PDE 4 inhibitors: by virtue of their ability to elevate cAMP content in leukocytes and airway smooth muscle, PDE 4 inhibitors may posses anti-inflammatory and bronchodilator activities.
Current PDE inhibitors used in treating inflammation and as bronchodilators, drugs like theophylline and pentoxyfyllin, inhibit PDE isozymes indiscriminently in all tissues These compounds exhibit side effects, apparently because they non-selectively inhibit all or most PDE isozyme classes in all tissues. This is a consideration in assessing the therapeutic profile of these compounds. The targeted disease state may be effectively treated by such compounds, but unwanted secondary effects may be exhibited which, if they could be avoided or minimized, would increase the overall therapeutic effect of this approach to treating certain disease states. Taken collectively, this information suggests that the side effects associated with the use of standard non-selective PDE inhibitors might be reduced by targeting novel isozyme-selective inhibitors for the predominant PDE in the tissue or cell of interest. Although in theory isozyme-selective PDE inhibitors should represent an improvement over non-selective inhibitors, the selective inhibitors tested to date are not devoid of side effects produced as an extension of inhibiting the isozyme of interest in an inappropriate or not-targeted tissue. For example, clinical studies with the selective PDE 4 inhibitor rolipram, which was being developed as an antidepressant, indicate it has psychotropic activity and produces gastrointestinal effects, e.g., pyrosis, nausea and emesis. Indications are that side effects of denbufylline, another PDE 4 inhibitor targeted for the treatment of multi-infarct dementia, may include pyrosis, nausea and emesis as well. These side effects are thought to occur as a result of inhibiting PDE 4 in specific areas of the CNS and gastrointestinal system.
In 1986, Schneider and colleagues described the presence and characteristics of high affinity, stereoselective [3H]-rolipram binding sites in rat brain homogenates. Although it was assumed that these binding sites represented the catalytic site of the rat brain xe2x80x9cnon-calmodulin-dependent, cAMP phosphodiesterasexe2x80x9d (i.e. PDE 4), a striking anomaly was apparent in the data. Under similar albeit not identical experimental conditions, data showed rolipram had a Kd=1 nM, whereas it inhibited rat brain PDE 4 activity with a Ki=1 xcexcM. Thus, there was a 1000-fold difference in the affinity of rolipram for the binding site versus its affect on catalytic activity. Although comprehensive structure activity relationships (SARs) for PDE inhibition and competition for [3H]-rolipram binding were not established, the substantial difference in the potency of rolipram as a PDE 4 inhibitor compared with its potency at the binding site seemed to question the validity of the assumption that both activities were contained within the same molecular locus.
Because of this conundrum, several studies were initiated. One sought to determine whether rolipram""s high affinity binding site existed on the same protein as the cAMP catalytic site. Another study sought to determine whether or not the SAR for inhibition of PDE 4 was the same as the SAR for competition with the high affinity rolipram binding site. A third study undertook to try and elucidate what biological significance, if any, there might be in these findings, particularly as it might relate to developing new drug therapies.
As data were collected from several assays, it became apparent that there are at least two binding forms on human monocyte recombinant PDE 4 (hPDE 4) at which inhibitors bind. One explanation for these observations is that hPDE 4 exists in two distinct forms. One binds the likes of rolipram and denbufylline with a high affinity while the other binds these compounds with a low affinity. Herein we distinguish these forms by referring to them as the high affinity rolipram binding form (HPDE 4) and the low affinity rolipram binding form (LPDE 4).
The importance of this finding lies in the discovery that compounds which potently compete for the high affinity rolipram binding form (HPDE 4) have more side effects or more intense side effects than those which more potently compete with the LPDE 4 (low affinity rolipram binding form). Further data indicate that compounds can be targeted to the low affinity binding form of PDE 4 and that this form is distinct from the binding form for which rolipram is a high affinity binder. Distinct SARs were found to exist for inhibitors acting at the high affinity rolipram binding form versus the low affinity rolipram binding form. In addition, these two forms appear to have different functional roles. Thus compounds that interacted with the low affinity rolipram binding form appear to have anti-inflammatory activity, whereas those that interact with the high affinity rolipram binding form produce side effects or exhibit more intensely those side effects.
There is no clear explanation for these findings. However, it is proposed the PDE 4 can exist in two distinct tertiary or quaternary states. Both forms are believed to be catalytically active. Rolipram binds to one catalytic site of one form with a high affinity, defined herein as having a Ki less than 10 nanomolar, and to the other form with a low affinity, defined here as having a Ki of greater than 100 nanomolar.
A useful consequence of these findings is that it is now possible to identify compounds which preferentially inhibit cAMP catalytic activity where the enzyme is in the form that binds rolipram with a low affinity, thereby reducing the side effects which apparently are linked to inhibiting the form which binds rolipram with a high affinity.
Chronic obstructive pulmonary disease (COPD) is an umbrella term frequently used to describe two conditions of fixed airways disease, chronic bronchitis and emphysema. Chronic bronchitis and emphysema are most commonly caused by smoking; approximately 90% of patients with COPD are or were smokers. Although approximately 50% of smokers develop chronic bronchitis, only 15% of smokers develop disabling airflow obstruction. Certain other mammals, particularly horses, suffer from COPD as well.
The airflow obstruction associated with COPD is progressive,.may be accompanied by airway hyperreactivity, and may be partially reversible. Non-specific airway hyper-responsiveness may also play a role in the development of COPD and may be predictive of an accelerated rate of decline in lung function in smokers.
COPD is a significant cause of death and disability. It is currently the fourth leading cause of death in the United States and Europe. Treatment guidelines advocate early detection and implementation of smoking cessation programs to help reduce morbidity and mortality due to the disease. However, early detection and diagnosis has been difficult for a number of reasons.
COPD takes years to develop and smokers often deny any ill effects from smoking, attributing the early warning signs of increased breathlessness as a sign of age. Similarly, acute episodes of bronchitis often are not recognized by the general practitioner as early signs of COPD. Many patients exhibit features of more than one disease (e.g. chronic bronchitis or asthmatic bronchitis) making precise diagnosis a challenge, particularly in early disease. Also, many patients do not seek medical help until they are experiencing more severe symptoms associated with reduced lung function, such as dyspnea, persistent cough, and sputum production. As a consequence, the vast majority of patients are not diagnosed or treated until they are in a more advanced stage of disease.
This invention thus provides a superior therapeutic index vis-à-vis COPD versus side effects.
This invention relates to a method for the prophylaxis of or treating COPD while minimizing gastrointestinal and psychotropic effects, which method comprises administering to a subject in need thereof an effective amount of a compound having an IC50 ratio of about 0.1 or greater as regards the IC50 for PDE 4 catalytic form which binds rolipram with a high affinity divided by the IC50 for the form which binds rolipram with a low affinity.
COPD is characterized by a chronic inflammatory process in the lung marked by in increase in the activation and/or number of alveolar macrophages, CD8+T-cells and neutrophils. Most notably with respect to the therapy of COPD is the ability to alter the trafficking and activation of neutrophils. The neutrophil is believed to play a central role in the pathophysiology of COPD. Neutrophil activation results in the release of a number of inflammatory mediators and proteinases, most importantly neutrophil elastase which contributes to the progressive fibrosis, airway stenosis and destruction of the lung parenchyma, leading to an accelerated decline in airway function. Neutrophil elastase is also a powerful mucus secretagogue and thus may contribute to the characteristic mucus hypersecretion that characterizes COPD. The compounds of this invention have marked effects on neutrophil activity, inhibiting neutrophil chemotaxis and degranulation in vitro. In animal models, the instant compounds reduce neutrophil extravasation from the circulation, pulmonary sequestration and the edematous responses to a number inflammatory insults in vivo.
Additional activities that may contribute to the therapeutic activity of PDE4 inhibitors in COPD include bronchodilation and modulation of pulmonary neuronal activity. Although the degree of reversibility of reduced airway flow is low in COPD, a small increase may have an acute positive effect, as well as a gradual reduction in the slope of the decline which may result in a profound effect on quality of life for COPD patients. The ability of inhaled muscarinc antagonists to produce clinically meaningful improvements in pulmonary function in COPD, at least acutely, suggest that a large component of the reversible airways obstruction in this disease is associated with a dysregulation of pulmonary nerves. Although not studied in detail as yet, PDE4 inhibitors may also modulate the activity of airway epithelial cells, a rich source of proinflammatory mediators that are released upon environmental insult (e.g., smoke), and inhibit vascular smooth muscle hyperplasia, a structural change in end stage COPD that is associated with right heart failure.
For purposes of this invention, the cAMP catalytic site which binds rolipram with a low affinity is denominated the xe2x80x9clow affinityxe2x80x9d binding site (LPDE 4) and the other form of this catalytic site which binds rolipram with a high affinity is denominated the xe2x80x9chigh affinityxe2x80x9d binding site (HPDE 4).
Initial experiments were conducted to establish and validate a [3H]-rolipram binding assay. Details of this work are given in Example 1 below.
To determine whether both the high affinity binding activity and the low affinity binding activity resided in the same gene product, yeast were transformed by known methods and the expression of recombinant PDE 4 was followed over a 6 hour fermentation period. Western blot analysis using an antibody directed against PDE 4 indicated that the amount of PDE 4 expressed increased with time, reaching a maximum after 3 hour of growth. In addition, greater than 90% of the immunoreactive product was in the high speed (100,000xc3x97g) supernatant of yeast lysates. [3H]R-(xe2x88x92)-Rolipram binding and PDE activity were monitored along with protein expression. PDE 4 activity was co-expressed with rolipram binding activity, indicating that both functions exist on the same gene product. Similar to results with the Western plot analysis, greater than 85% of the rolipram-inhibitable PDE activity and [3H]-rolipram binding activity was found to be present in the yeast supernatant fraction.
Overall, most of the recombinant PDE 4 expressed in this system exists as LPDE 4 and only a small fraction as HPDE 4. Consequently, inhibition of recombinant PDE 4 catalytic activity primarily reflects the actions of compounds at LPDE 4. Inhibition of PDE 4 catalytic activity can thus be used as an index of the potency of compounds at LPDE 4. The potency of compounds at HPDE 4 can be assessed by examining their ability to compete for [3H]R-(xe2x88x92)-rolipram. To develop structure-activity relationships (SARs) for both the low affinity and high affinity rolipram binding sites, the potencies of selected compounds were determined in two assay systems. Results from experiments using standard compounds were tabulated. As expected, certain compounds were clearly more potent in competing with [3H]-rolipram at the site for which rolipram demonstrated high affinity binding as compared with the other site, the one at which rolipram is a low affinity binder. SAR correlation between high affinity binding and low affinity binding was poor and it was concluded that the SAR for inhibition of high affinity [3H]-rolipram binding was distinct from the SAR for binding to the low affinity rolipram binding site. Table I provides results from this SAR work.
Denbufylline is 7-acetonyl, 1,3-dibutylxanthine made by SmithKline Beecham. Papaverine is 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline. Trequinsin is 2,3,6,7-tetrahydro-2-(mesitylimino)-9,10-dimethoxy-3-methyl-4H-primido[6,1-a]isoquinoline-4-one. Dipyrimadole is the generic name for 2,2xe2x80x2,2xe2x80x3,2xe2x80x2xe2x80x3-[(4,8-dipiperidinopyrimido[5,4-d]pyrimidine-2-6-diylidinitrilo]tetraethanol.
These results illustrate that some compounds can selectively inhibit the so called low affinity form as compared with the high affinity form, and vice versa. The significance of this finding is that it is feasible to minimize side effects by designing or choosing compounds which selectively (preferentially) inhibit one site thereby affecting the desired response to the exclusion of another, unwanted, response, or at least to minimize the non-targeted response to a degree where it is not interfering with the intended therapy to an unacceptable degree.
Notwithstanding this work, we have not defined the basis for the disparate SARs for high affinity rolipram binding and low affinity rolipram binding in the PDE 4 isozyme,. However it has been discovered that if a compound exhibits an IC50 ratio of about 0.1 or greater, calculated as the ratio of the IC50 for high affinity rolipram binding form divided by the IC50 for the form which binds rolipram with a low affinity, it will have an acceptable therapeutic index. That is, one can now successfully treat a variety of immune and inflammatory diseases while not affecting other physiological phenomena at all or to an unacceptable degree. Herein the most preferred embodiment is inhibiting the low affinity rolipram binding site as a means for treating inflammatory and allergic diseases.
This invention covers those compounds which have an IC50 ratio (high/low binding) of about 0.1 or greater. This includes any and all compounds which are PDE 4 inhibitors as per the test set our herein, and which demonstrate in these, or similar assays, a ratio within the defined range; of particular interest are those compounds which are not in the public domain and/or not tested as or known to be PDE 4 inhibitors prior to the filing date of this application.
A preferred technique for selecting useful compounds is that of determining one having an IC50 ratio of about 0.1 or greater ratio of the IC50 value for competing with the binding of 1 nM of [3H]R-rolipram to a form of PDE 4 which binds rolipram with a high affinity over the IC50 value for inhibiting the PDE 4 catalytic activity of a form which binds rolipram with a low affinity using 1 xcexcM[3H]-cAMP as the substrate.
Examples of compounds which meet the IC50 ratio standard are given in Table 1 above as well as U.S. Pat. No. 5,448,686; U.S. Pat. No. 5,605,923; and U.S. Pat. No. 5,552,438. Each of these applications is incorporated herein by reference in full as if set out in this document.
Preferred compounds of this invention are those which have an IC50 ratio of greater than 0.5; and particularly those compounds having a ratio of greater than 1.0. Compounds such as cis-[4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylate], 2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one, and cis-[4-cyano-4-(3-cyclopropyimethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol] are examples of structures which bind preferentially to the low affinity binding site and which have an IC50 ratio of 0.1 or greater.
The present compounds and pharmaceutically acceptable salts may be administered in a standard manner for the treatment of the indicated diseases, for example orally, parenterally, sub-lingually, dermally, transdermally, rectally, via inhalation or via buccal administration. A controlled-release preparation can also be utilized.
The present compounds and pharmaceutically acceptable salts which are active when given orally can be formulated as syrups, tablets, capsules, controlled-release preparation or lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil, olive oil, glycerine or water with a flavoring or coloring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils, and are incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of a compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.
Typical dermal and transdermal formulations comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.
Preferably the composition is in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer a single dose.
Each dosage unit for oral administration contains suitably from 0.3 mg to 60 mg/Kg, and preferably from 1 mg to 30 mg/Kg of a compound or a pharmaceutically acceptable salt thereof. Preferred doses include 10 mg and 15 mg/Kg for treating COPD. Each dosage unit for parenteral administration contains suitably from 0.1 mg to 100 mg/Kg, of the compound or a pharmaceutically acceptable salt thereof. Each dosage unit for intranasal administration contains suitably 1-400 mg and preferably 10 to 200 mg per person. A topical formulation contains suitably 0.01 to 5.0% of a present compound.
The active ingredient may be administered from 1 to 6 times a day, sufficient to exhibit the desired activity. Preferably, the active ingredient is administered about once or twice a day, more preferably twice a day.
No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention.